Revealing the Full Proteoform Landscape with the Enhanced Power of Top-down Mass Spectrometry

Proteomics, the large-scale study of proteins, depends on mass spectrometry (MS) as the key tool for analyzing protein structure, modifications, and function. Traditionally, this has involved approaches that require breaking proteins into their building blocks before analysis. Much like early DNA and RNA sequencing methods that relied on sequencing fragmented molecules and assembling the results, “bottom-up” proteomics digests proteins into peptides and then infers the protein’s sequence and any post-translational modifications (PTMs) from these fragments. While powerful and suitable for high-throughput analysis, this peptide-centric approach does not reveal important information about how modifications and sequence variations coexist on the same intact protein.

Recent advances in “top-down” proteomics are offering new opportunities for researchers to study intact proteins directly. Scientists can determine the complete amino acid sequence, locate and identify post-translational modifications (PTMs), and characterize structure—all in a single experiment without up-front digestion. Though still technically challenging, top-down proteomics offers a new window into the true complexity of protein species and promises to complement, rather than replace, traditional bottom-up methods. Here, we explore these developments and what the future might hold.

The Rise of the Proteoform Perspective

The Human Genome Project, whichdetermined the DNA base-pair sequence and identified and mapped all human genes, revealed that the number of genesis far smaller than once assumed (~ 20,000), prompting the scientific community to recognize that biological complexity in humans largely arises not from gene number but from variation at the protein level (1). This variation includes genetic differences, alternative RNA splicing, and the wide array of PTMs, resulting in distinct molecular forms of proteins, termed proteoforms, that drive different cellular functions. The belief that understanding and accurately cataloguing these proteoforms is central to deciphering the functional output of the genome has become widely adopted among leading researchers (2,3).

Top-down proteomics approaches center around analyzing intact proteins in their full form to retain information about how modifications and sequence variations coexist, enabling the comprehensive identification and study of different proteoforms. Characterizing proteoforms reveals protein diversity, and gives clear insights into the complexity of protein function and regulation that may be missed when studying only peptide fragments with bottom-up methods. Analysis of whole proteins without digestion therefore provides a holistic view of the proteome, which could enable researchers to decipher protein function, uncover disease mechanisms, and advance precision medicine (4).

In practice, top-down proteomics involves introducing intact protein ions into the mass spectrometer where they undergo fragmentation in the gas phase. This fragmentation produces ions that provide information across the entire amino acid sequence, enabling precise localization of PTMs within the context of the complete protein structure. This method allows for sequence determination and modification mapping in a single experiment, without the need for additional steps.

Although the approach provides detailed information, it still presents some challenges. The complexity and size of intact proteins result in more complex mass spectra that require sophisticated software and computational methods for analysis. In addition, effective front-end separation and sample preparation techniques are necessary to reduce sample complexity and improve detection, which remain areas of ongoing development. These factors currently limit the widespread adoption of top-down proteomics.

Bottom-up Proteomics: Strengths and Limitations

Bottom-up proteomics is the most widely used approach in protein analysis and involves enzymatically digesting proteins into smaller peptide fragments before mass spectrometry (MS) analysis. The typical workflow starts with protein digestion, often using proteases like trypsin, followed by liquid chromatography separation and tandem MS (LC–MS/MS) to identify peptides. From peptide-level data, protein inference is then performed to reconstruct which proteins were present in the original sample.

Bottom-up proteomics can be high throughput, meaning it efficiently handles large and complex biological samples, enabling the identification and quantification of thousands of proteins simultaneously. The maturity of bottom-up proteomics is evident in its well-established protocols, bioinformatics pipelines, and extensive databases, which allow for reproducible and standardized analysis. Bottom-up methods also exhibit high sensitivity, which makes them effective for detecting low-abundance proteins as a result of the favorable ionization and fragmentation properties of peptides.

The bottom-up approach, however, has inherent limitations. The process of digesting proteins into peptides destroys the connectivity information of the original intact protein molecule. As a result, bottom-up proteomics can’t determine which combinations of PTMs coexist on the same protein molecule, leading to loss of detailed proteoform information. Identifying PTMs is a particular challenge as, although PTM sites can often be predicted based on their peptide data, their exact location within a native protein remains unclear. It is also difficult to distinguish multiple modifications on the same protein molecule, such as a phosphorylation at two or more distinct sites, because peptides represent only segments of the protein and are disconnected from each other. Additionally, bottom-up proteomics struggles to directly determine the composition and linkage structure of glycans, typically meaning that supplementary experiments or indirect inference are needed to characterize glycosylation. In summary, while bottom-up proteomics is highly effective for broad protein identification and quantification, it does not preserve the molecular context of intact proteins in their complex modification patterns, highlighting the complementary role of top-down proteomics, which seeks to analyze proteins in their intact forms.

What’s New?

Researchers have long understood that, to make top-down proteomics a practical reality, significant advances in several key areas are required. These include increased sensitivity to detect intact proteins at biologically relevant concentrations, high-speed multimodal fragmentation analysis to generate rich and informative fragment ion data, and high-performance ionization techniques capable of efficiently transferring large, often fragile protein ions into the gas phase. Recent developments in integrated mass spectrometry platforms that combine ion mobility separation with flexible electron- and collision-based fragmentation have contributed significantly to this progress. These systems enable detailed sequencing of intact proteins and precise localization of PTMs within a single analysis. Advances in computational tools now support automated processing of complex spectra, improving identification confidence and throughput. Continued technological refinement to help with front-end sample preparation and to handle the large data volumes generated is important to help expand the accessibility and routine use of top-down proteomics in biological and clinical research.

Pioneers of Top-down Proteomics

Multi-level Analysis of Protein Complexes Using Native MS and Top-down Proteomics

Abraham Oluwole and colleagues at the University of Oxford have demonstrated the advantages of integrating native mass spectrometry with advanced top-down sequencing and PTM characterization to study protein complexes critical for cellular function and drug development (see Figure 1 [5]) (6). Their work highlights how combining native MS with multi-stage fragmentation (MSⁿ) and ion enrichment techniques offers a direct and detailed view of protein oligomeric states, subunit interactions, and PTM patterns, as well as their binding to ligands or inhibitors. This integrated approach was effectively applied to analyze the β-barrel assembly machinery (Bam), a heteropentameric protein complex that facilitates the insertion of β-barrel proteins into the outer membrane of Gram-negative bacteria. By preserving native protein interactions and performing detailed fragmentation-based sequencing, this methodology provides critical structural and functional insights into complex protein assemblies that are difficult to access with conventional mass spectrometry methods.

Decrypting Histone Modifications

Ole N. Jensen and colleagues at the Danish National Mass Spectrometry Platform have highlighted the crucial role that detailed characterization of histone PTMs plays in understanding gene regulation and its impact on diseases such as cancer, neurodegenerative disorders, cardiovascular diseases, and metabolic conditions (7).Histones are fundamental for DNA packaging, and their diverse PTMs modulate chromatin structure and function, making accurate and comprehensive mapping of these modifications essential. Due to the high variability and complexity of modification sites within histone proteins, standard approaches often fall short in fully resolving PTM patterns. To address this, Jensen’s team employs advanced multimodal fragmentation techniques combined with sophisticated computational algorithms that enable high-confidence positional assignment of PTMs directly on intact histones. Using a combination of electron-based dissociation methods and collision-induced dissociation (CID), their approach achieves extensive sequence coverage of around 70% to 80% with individual electron-based methods and approximately 40% with CID (See Figure 2 [5]). Notably, integrating multiple fragmentation types on highly charged histone ions provides complete sequence coverage, allowing unambiguous localization of modifications such as acetylation on H3.1K14. This comprehensive top-down proteomics strategy facilitates detailed proteoform-level insights into histone modification landscapes, which are key to deciphering disease mechanisms and epigenetic regulation.

A Proteoformics Future

While bottom-up proteomics maintains its role in broad protein identification and quantification, top-down proteomics has shown its potential as a complementary approach, which opens the door to a more detailed view of protein complexity. Together, integrated hierarchical workflows that combine both approaches can maximize biological insight.

Looking ahead, advances in front-end protein separation and the speed and sensitivity of MS instruments are expected to broaden the accessibility of top-down proteomics, with democratization anticipated in the next decade. This proteoform-resolved perspective has the potential to transform biomedical research and industry applications alike.

Embracing emerging technologies and methodologies in top-down proteomics will be important for the next generation of proteomics research. As a powerful and complementary strategy, top-down proteomics will support more precise understanding of proteome dynamics. Preparing the research community for this shift will facilitate its adoption and, ultimately, accelerate scientific discovery through practical application.

References

(1) National Human Genome Research Institute. 2001: First Draft of the Human Genome Sequence Released. https://www.genome.gov/25520483/online-education-kit-2001-first-draft-of-the-human-genome-sequence-released (accessed 2025-07-31).

(2) Smith, L.; Kelleher, N.; The Consortium for Top Down Proteomics. Proteoform: A Single Term Describing Protein Complexity. Nat. Methods 2013, 10, 186–187. DOI: 10.1038/nmeth.2369

(3) Schaffer, L. V.; Millikin, R. J.; Shortreed, M. R.; Scalf, M.; Smith, L. M. Improving Proteoform Identifications in Complex Systems Through Integration of Bottom-Up and Top-Down Data. J. Proteome Res. 2020, 19 (8), 3510–3517. DOI: 10.1021/acs.jproteome.0c00332

(4) Roberts, D. S. Loo, J. A.; Tsybin, Y. O.; et al. Top-down Proteomics. Nat. Rev. Methods Primers 2024, 4 (1), 38. DOI: 10.1038/s43586-024-00318-2

(5) Bruker. timsOmni. https://www.bruker.com/en/products-and-solutions/mass-spectrometry/timstof/timsomni.html (accessed 2025-08-07)

(6) Oluwole, A.; Shutin, D.; Bolla, J. R. Mass Spectrometry of Intact Membrane Proteins: Shifting Towards a More Native-like Context. Essays Biochem. 2023, 67 (2), 201–213. DOI: 10.1042/EBC20220169

(7) Berthias, F.; Bilgin, N.; Mecinović, J.; Jensen, O. Top‐down Ion Mobility/Mass Spectrometry Reveals Enzyme Specificity: Separation and Sequencing of Isomeric Proteoforms. PROTEOMICS 2024, 24. 2200471. DOI: 10.1002/pmic.202200471